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    Home > Biochemistry News > Biotechnology News > Gene mutation detection technology.

    Gene mutation detection technology.

    • Last Update: 2020-10-21
    • Source: Internet
    • Author: User
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    methods for analyzing unknown mutations .1.RNA enzyme A cutting (RnaseAcleavage
    Under certain conditions, the mismatched base in the isogenetic double-stranded nucleic acid molecule RNA: RNA or RNA:
    DNA
    can be cut by RNaseA, and the cutting product can be isolated by running denatured glue. RNA probes can be obtained by cloning corresponding DNA fragments into vectors containing SP6 or T7 initiator, and RNaseA's cutting efficiency at misaled places is low or not cut when the misalgenic base is radon, and when the misaltaged base is nipples, the cutting efficiency is high. Therefore, if only one chain of the DNA being examined is analyzed, the mutation detection rate is only 30%, and if the justice chain and anthotic chain of the DNA being examined are analyzed at the same time, the efficiency can reach 70%. Despite the limitations of RNaseA cutting, the
    PCR
    product needs to be cloned to the expression vector to use the
    isotope
    , thus increasing the complexity of the operation, but because it can detect fragments of 1 to 2kb, and can determine the mutation location, and do not need to use harmful
    chemical reagents
    , it is still frequently used.. 2. Degeneration gradient gel electrophoresis (DGGE)
    The principle of this method is: double-stranded DNA molecules in a certain denaturing agent concentration of gel electrophoresis, will occur at a certain time a partial unchain, resulting in a decrease in electrophoresis migration rate, when the normal DNA molecules and the DNA molecules of the protruding master, even if there is only one base pair difference, will occur at different times part of the unchain, and thus be separated into two
    3. Hybrid double-stranded analysis (HA)
    Heterogeneous hybrid double-stranded DNA formed by mutant and wild DNA forms a bulge at its misalignment, and when electrophoresis occurs in non-denatured gum, a different migration rate is produced than the corresponding congenerous double-stranded DNA, so that the two are divided. The principle of this method is similar to single-chain structure polymorphism (SSCP), except that SS-CP is separated by a single chain, while HA is separated by a double chain. HA method is simple and rapid, but only applicable to 200 to 300pb fragments, and can not determine the mutation location, detection rate is only
    about 80%, it is suggested that the HA method and SSCP method combined use, may increase the detection

    rate to nearly 100%.
    chemical cutting mismatch is a mutation detection technique developed on the basis of Maxam-Gilbert sequencing, in DNA: DNA or DNA: RNA heterogenous hybrid double-stranded nucleic acid molecules, mismatched C can be cut by hydroxylamine and piperidine, mismatched T can be cut by Osmiumtetroxide, cutting products run adhesive electrophoresis can determine whether there is a mutation.
    As long as the operation is done well, non-specific cutting will not occur, if both the justice chain and the antisexual chain are analyzed, the detection rate can reach 100%, the use of fluorescence detection system will greatly enhance the sensitivity of the method, so that one of the ten cells can be detected mutant cells. The biggest advantage of this method is that it can analyze fragments as long as 2kb and determine the mutation location, the disadvantage is that it is many steps, time-taking, and needs to be exposed to toxic chemicals.
    5. Carbodiimide (CDI)
    is similar to the CCM method, where CDI is able to modify misaligned G and T, and when DNA synthesis of CDI-modified double-stranded DNA is performed with labeled citations, the extension is terminated at the modified base, and the synthetic product runs denatured polyacrylamide gel electrophoresis to detect mutations. The greatest advantage of this method is also the ability to analyze fragments of 1 to 2kb, mutation detection rate of 100%, can determine the location of mutations, and CDI is a non-toxic substance, it has been reported that the method has detected a point mutation in 7.2kb DNA fragments, but when there are multiple mutations in a DNA fragment, the method does not apply.
    6. EnzymeMismatchCleavage, EMC) .EMC is a similar method to RNaseA cutting, T4 nucleic acid incision enzyme is a decomposition enzyme (resolvase), can identify 12 mismatched bases and cut near the mismatched base, enzyme cutting products run denatured or neutral glue can detect whether there is a mutation. Because the method can analyze DNA: DNA heterogeneous hybrid molecules, thus omitting the step of in-body transcription, its longest detection fragment can reach 1.5kb, the disadvantage of the method is the existence of non-specific cutting phenomenon, and some misalmuted base recognition is not 100%, so each test must have an internal control.
    7. Single-stranded composition polymorphism (Single-StrandCon-formational Polymorphism, SSCP)
    The principle of this approach is that the single-stranded DNA forms a secondary structure under neutral conditions, which depends on its base composition, even if a base is different, and causes different secondary structures and causes electrophoretic migration rates
    8.
    (Cleavage Frag-mentLength Polymorphism CFLP) CFLP technology is a gene mutation detection technology developed on the basis of restrictive enzyme-cut fragment length polymorphism (RELP) and SSCP, based on the principle that the single chain formed by double-stranded DNA denaturation forms a secondary structure (containing multiple folded hair clamping structures) under neutral conditions, as in the case of SSCP, this secondary structure depends on DNA.
    nucleotide
    composition and sequence, even if there is a base difference, will form a different number of folding hairpin structure, and CleavaseI exocessase can identify this hair clip structure, and in the vicinity of the structure to cut, cutting products Run-denatured polyacrylamide gel detection, mutant DNA will appear different from normal control enzyme-cutting map
    9.DNA sequencing
    mutations detected by various mutation detection techniques must eventually be sequenced by sequencing To determine the mutation type and mutation location, and the sequencing method to detect mutation efficiency of 100%, based on this, some researchers proposed that some smaller, less exons of the gene mutation detection directly with sequencing, such as CMTX CX Mutation analysis of the 32 genes, but the method requires more labor and exposure to isotopes, and the DNA autosequencer introduced by PEABD uses four-color fluorescent markers instead of the original hand-sequenced isotope markers. And sequencing and analysis of data time is also greatly shortened, but the disadvantage is expensive, so some larger, more exons of genes should not be used sequencing method to directly detect mutations, but also not applicable to clinical detection of a large number of specimens, in addition, sequencing can not be regarded as a gold standard for mutation detection, hetero-mutation, glue compression, the existence of GC rich areas and other issues make it difficult to obtain accurate data through a sequencing.
    10. DideoxyFinger-printing, ddF
    Sensitivity of SSCP detection method is affected by electrophoresis temperature, PCR fragment size and other factors The method of direct DNA sequencing is time-timed and costly, so some researchers combine SSCP and Sanger double deoxygenation sequencing in practice to form a new mutation detection method, ddF. The genetic principle of this method is: after the recovery and purification of PCR products with two citants to do Sanger double deoxygenation sequencing reaction, but the difference is only added a double deoxygenation terminator, the reaction product running neutral polyacrylamide gel electrophoresis if there is a mutation, in the patient's electrophoresis map will be lost or obtained a band or at least one band migration rate will change. In the ddF method, the migration rate of the DNA chain depends not only on its secondary structure, but also on its size, making it more sensitive than the SS-CP method.
    MartincicD and others have compared the SSCP method with the ddF method and found that ddF can detect all 10 known p53 tumor mutations, while the SSCP method can detect only 6 of the 10 mutations. The sensitivity of the ddF method is not affected by electrophoresis temperature and can detect 6 of the 10 mutations. The sensitivity of the ddF method is not affected by the electrophoresis temperature, the length of the PCR product can be detected can reach nearly 500bp, and the method can also be relatively positioned for mutation. The main disadvantage of this method is the need to use isotopes, while the purity of the recovered PCR products is high, non-specific PCR products will seriously affect the analysis of the final results.
    11. Misalmed joint protein detection
    A DNA misalmed joint protein isolated from Ecoli can join DNA at the misalmed base in heterogenous hybrid double-stranded DNA, and the jointed product runs the sequencer glue to detect mutations through changes in electrophoretic migration rates. The advantage of this method is that the operation is simple, can handle a large number of samples at once, but it is reported that this DNA misalmed binding protein to a single base misalmed joint is not as strong as the joint of several base misalmouts, and this protein also has a certain binding to the non-misalmation area leading to a higher background, recently some researchers suggested that the combination of several DNA misalty joint proteins will improve the accuracy of mutation detection.
    12. Denaturation of highly effective liquid phase
    chromatography
    detection (Denaturing High Performance LiguldChromatography DHPLC)
    DHPLC technology is a The new hybrid double-stranded mutation detection technology developed on the basis of DGGE is similar to that of DGE, i.e. the mismatched heterogenous hybrid double-stranded DNA and the perfectly matched hoseed double-stranded DNA can be separated by HPLC under partially denatured conditions. DHPLC technology, which has been used to compare DNA sequencing, is being used by a team of researchers in Stanford to detect disease-related genetic mutations. Compared with the traditional hybrid double-stranded analysis technology, the technology takes a short time, the analysis of a sample generally only takes 5 minutes, does not need to use radioisotopes, the degree of automation is high, but needs to buy another HPLC instrument. Although the technology is currently mainly used to detect 200 to 300bp-sized DNA fragments, long DNA fragments have not been reported, but researchers are optimistic.
    13.DNA Chip Technology (DNAchip)
    .DNA chip technology is a new technology developed since the 1990s, which combines integrated circuit computer, semiconductor, laser co-aggregation scanning, fluorescent marker probe and oligonucleotide DNA synthesis technology, fully embodies biological technology and other important roles, such as gene positioning, DNA sequencing, physical map and genetic map construction, etc. The nucleotide DNA is arranged on an integrated circuit board, overlapping one base with each other, and covering the entire gene to be tested, the normal and mutated DNA of fluorescence markers are interbred with two DNA chips, and due to at least one base difference, the normal DNA and mutant DNA will be given different hybrid maps, and fluorescent signals from the two DNA molecules can be determined by focusing
    microscopes
    respectively to determine if there is a mutation..
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